Saliva is a readily accessible biofluid that is important for the overall health, aiding in the chewing, swallowing, and tasting of food as well as the regulation mouth flora. As a first step to determining and understanding the human saliva metabolome, we have measured salivary metabolite concentrations under a variety of conditions in a healthy population with reasonably good oral hygiene. Using (1)H NMR spectroscopy, metabolite concentrations were measured in resting (basal) and stimulated saliva from the same subject and compared in a cohort of healthy male non-smoking subjects (n = 62). Almost all metabolites were higher in the unstimulated saliva when compared to the stimulated saliva. Comparison of the salivary metabolite profile of male smokers and non-smokers (n = 46) revealed citrate, lactate, pyruvate, and sucrose to be higher and formate to be lower in concentration in smokers compared with non-smokers (p < 0.05). Gender differences were also investigated (n = 40), and acetate, formate, glycine, lactate, methanol, propionate, propylene glycol, pyruvate, succinate, and taurine were significantly higher in concentration in male saliva compared to female saliva (p < 0.05). These results show that differences between male and female, stimulated and unstimulated, as well as smoking status may be observed in the salivary metabolome.